Growth hormone-releasing factor analogs

ABSTRACT

Human Growth Hormone-Releasing Factor (hGRF) analogs having the sequence [X 3 , Y 8 , Z 25 , Nle 27  ]-hGRF(1-A)--B, wherein X, Y and Z are selected from the group consisting of Asn and Asp, A has a value from 29-44, and B is NH 2  or OH are synthesized and administered to animals to stimulate the release of Growth Hormone (GH).

.Iadd.This application is a reissue of U.S. Pat. No. 4,801,456..Iaddend.

This invention relates generally to human growth hormone-releasingfactor (hGRF) analogs and particularly to hGRF analogs having thesequence: [X³, Y⁸, Z²⁵, Nle²⁷ ]- hGRF(1-A)-B, wherein X, Y, and Z areselected from the group consisting of Asn and Asp, A has a value from29-44, and B is NH₂ or OH.

BACKGROUND OF THE INVENTION

Human Growth hormone-releasing Factor (hGRF) is a 44 amino acid peptidehaving growth hormone (GH) releasing activity as reported by Guilleminet al., 218, Science 585 (1982). hGRF is usually isolated frompancreatic human tumor cells (hpGRF). hpGRF has the structureH-Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Gly-Ala-Arg-Ala-Arg-Leu-NH₂.Since the initial discovery of hGRF, several peptides, designated hereinas analogs, having deleted, substituted or otherwise modified sequenceshave been reported to have GH releasing activity. Rivier et al., 300Nature 276 (1982) reported a peptide that terminates as a freecarboxylic acid and differs from hGRF by the absence of the C-terminaltetrapeptide amide -Arg-Ala-Arg-Leu-NH₂. Rivier et al. tested a numberof shortened GRF analogs and reported that, when compared in vitro withthe parent hpGRF(1-40)--OH, the following analogs exhibited similaractivity: hpGRF(1-29)--NH₂, hpGRF(1-32)--NH₂, hpGRF(1-39)--NH₂,hpGRF(1-40)--NH₂, and hpGRF(1-27)--NH₂.

Numerous synthetic GRF peptides and GRF analogs have been patented: U.S.Pat. No. 4,610,976 to Bohlen discloses 44 amino acid synthetic peptidesdescribed as extremely potent in stimulating the release of pituitary GHin mammals. These synthetic peptides, biologically active fragmentsthereof, analogs thereof, or nontoxic salts thereof can be dispersed ina pharmaceutically acceptable carrier and administered for diagnostic ortherapeutic purposes. The 44 amino acid polypeptide is believed to beporcine GRF. U.S. Pat. No. 4,605,643 to Bohlen discloses a 44 amino acidsynthetic polypeptide that is the replicate of the native GRF of thesheep hypothalmus. The peptide is extremely potent in stimulating therelease of GH in mammals. The patent states that the peptide,biologically active fragments thereof, analogs thereof, or nontoxicsalts thereof may be administered to animals for therapeutic ordiagnostic purposes. As examples of biologically active fragments,Bohlen states that fragments 34-43 residues in length, or even shorterfragments, e.g. oGRF (1-32) that retain an --OH or --NH₂ of theC-terminal and retain the desired biological activity are suitable. U.S.Pat. No. 4,595,676 to Spiess discloses the synthesis of rat hypothalamicGRF. A number of polypeptides, which have 44 amino acids, and are usefulin stimulating the release of GH in animals are disclosed. Referencealso is made to biologically active fragments of the polypeptides. U.S.Pat. No. 4,585,756 to Brazeau discloses a 44-residue polypeptideisolated from purified extracts of bovine hypothalami and useful forpromoting the growth of animals. Reference is made to biologicallyactive fragments thereof, including bGRF(1-40) and bGRF (1-37) orshorter fragments. U.S. Pat. No. 4,563,352 to Rivier describes thesynthesis of human pancreatic GRF and biologically active fragmentsthereof and provides synthetic peptides useful in stimulating therelease of pituitary GH in mammals. U.S. Pat. No. 4,562,175 to Changdiscloses a synthetic peptide GRF useful in growth inducingpharmaceutical compositions. The peptide is based on the structure ofhuman pancreatic GRF. The synthetic peptide differs from the naturalpeptide by comprising norleucine in place of methionine at position 27.Other analogous peptides susceptible to a similar modification also aredisclosed, including [D-Ala², Nle²⁷ ]-hpGRF(1-44)--NH₂ and [D-Ala²,Nle²⁷ ]ratGRF(1-43)--NH₂). U.S. Pat. No. 4,529,595 to Rivier disclosesanalogs of hpGRF useful in stimulating the release of pituitary GH inmammals. Biologically active fragments, said to generally extend fromthe N-terminal to a residue between positions 27 and 32, also aredisclosed. U.S. Pat. No. 4,528,190 to Vale provides syntheticpolypeptides, useful in stimulating the release of pituitary GH inanimals, which have resistance to enzymatic degradation in the body.U.S. Pat. No. 4,518,586 to Rivier describes a 44-amino acid syntheticpolypeptide in which any or all of the residues between the 29th and44th residues may be deleted. U.S. Pat. No. 4,517,181 to Ling disclosessynthetic porcine GRF peptides which promote the release of GH by thepituitary gland and teaches that deletions can be made beginning at thecarboxyl end of the peptide to create fragments that retain substantialportions of the potency of the peptide. U.S. Pat. No. 4,617,149discloses a class of 44-amino acid polypeptide analogs of hpGRF bearingsubstitutions of the amino acid at position 27. Other similar GRFanalogs are disclosed in U.S. Pat. Nos. 4,622,312 and 4,626,523.

Similarly, there have been many publications relating to GRF analogs:Ling et al., Synthesis and In Vitro Bioactivity of C-Terminal DeletedAnalogs of Human Growth Hormone-Releasing Factor, Biochem. Biophys. Res.Commun., 123(2), 854-861 (1984); Ling et al., Synthesis and In VitroBioactivity Human Growth Hormone-Releasing Factor Analogs Substituted atPosition-1, Biochem. Biophys. Res. Commun., 122(1), 304-310 (1984);Wehrenberg et al., In Vitro Biological Potency of Rat and Fragments,Biochem. Biophys. Res. Commun., 115(2), 525-530 (1984); Coy et al.,Structure-Activity Studies on the N-Terminal Region of, J. Med. Chem.,28, 181-185 (1985); and Lance et al., Super-Active Analogs (1-29)-Amide,Biochem. Biophys. Res. Commun., 119(1), 265-272 (1984).

Although numerous patents and publications relating to GRF analogs havebeen disclosed in the prior art, there exists a continuing need forsynthetic GRF analogs which stimulate the release of GH and induce thebeneficial effects associated therewith.

SUMMARY OF THE INVENTION

It is, therefore, an object of the present invention to provide humangrowth hormone-releasing factor (hGRF) analogs.

It is another object of the present invention to provide hGRF analogswhich stimulate the release of pituitary growth hormone (GH) in animals.

It is another object of the present invention to provide hGRF analogshaving substitutions at positions 3, 8, 25, and 27.

It is another object of the present invention to provide hGRF analogshaving various combinations of Asn and Asp at positions 3, 8, and 25.

It is a further object of the present invention to provide a compositioncontaining the hGRF analogs of the present invention suitable foradministration to animals to stimulate the release of pituitary GH.

These and other objects are achieved by synthesizing hGRF analogs havingthe sequence [X³, Y⁸, Z²⁵, Nle²⁷ ]-hGRF(1-A)--B, wherein X, Y, and Z areselected from the group consisting of Asn and Asp, A has a value from29-44, and B is NH₂ or OH, and administering the hGRF analogs to animalsto stimulate the release of GH and induce the beneficial effects ofincreased GH levels.

In the preferred embodiment, hGRF analogs having the sequence [X³, Y⁸,Z²⁵, Nle²⁷ ]-hGRF (1-31)--NH₂, wherein X, Y, and Z are selected from thegroup consisting of Asn and Asp, are synthesized and administered toanimals to stimulate the release of GH and induce the beneficial effectsof increased GH levels.

Other objects, advantages, and novel features of the present inventionwill become apparent from the following detailed description of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

The amino acids referred to herein are described by shorthanddesignations as follows:

    ______________________________________                                        Amino Acid Nomenclature                                                       Name             3-letter  1-letter                                           ______________________________________                                        Alanine          Ala       A                                                  Arginine         Arg       R                                                  Asparagine       Asn       N                                                  Aspartic Acid    Asp       D                                                  Cysteine         Cys       C                                                  Glutamic Acid    Glu       E                                                  Glutamine        Gln       Q                                                  Glycine          Gly       G                                                  Histidine        His       H                                                  Homoserine       Hse       --                                                 Isoleucine       Ile       I                                                  Leucine          Leu       L                                                  Lysine           Lys       K                                                  Methionine       Met       M                                                  Methionine       Met (O)   --                                                 sulfoxide                                                                     Methionine       Met (S-Me)                                                                              --                                                 methylsulfonium                                                               Norleucine       Nle       --                                                 Phenylalanine    Phe       F                                                  Proline          Pro       P                                                  Serine           Ser       S                                                  Threonine        Thr       T                                                  Tryptophan       Trp       W                                                  Tyrosine         Tyr       Y                                                  Valine           Val       V                                                  ______________________________________                                    

To simplify the nomenclature used to disclose the peptide sequences ofthe present invention, the following well-known shorthand notation wilbe used herein: [X³, Y⁸, Z²⁵, Nle²⁷ ]-hGRF(1-A)--B, wherein the (1-A)indicates that the peptide has the same sequence as the first A aminoacids of the original 44-residue hGRF; except for the amino acids atpositions 3, 8, 25, and 27 which have been replaced by amino acidsdesignated X, Y, Z, and Nle, respectively. For example, [Asn³, Asp⁸,Asn²⁵, Nle²⁷ ]-hGRF(1-31)--NH₂ indicates a peptide 31 amino acids longwhose sequence is the same as the first 31 amino acids of native hGRFexcept that Nle has been substituted for Met at position 27, Asn hasbeen substituted at position 25, Asp has been substituted at position 8,and Asn has been substituted at position 3 when read from the aminoterminal end of native GRF. The complete sequence, expressed using the 3letter code, for this amino acid would therefore beH-Tyr-Ala-Asn-Ala-Ile-Phe-Thr-Asp-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asn-Ile-Nle-Ser-Arg-Gln-Gln-NH₂.Similarly, [Asn³, Nle²⁷ ]-hGRF (1-31)--NH₂ indicates a peptide 31 aminoacids long, whose sequence is the same as the first 31 amino acids ofnative hGRF except that Nle has been substituted for Met at position 27and Asn has been substituted at position 3.

According to the present invention, human growth hormone-releasingfactor (hGRF) analogs having the sequence [X³, Y⁸, Z²⁵, Nle²⁷]-hGRF(1-A)--B, wherein X, Y, and Z are selected from the groupconsisting of Asn and Asp, A has a value from 29-44, and B is NH₂ or OH,are synthesized and used to stimulate the release of growth hormone (GH)in animals. The hGRF analogs of the present invention are peptideshaving the general sequence of hGRF but differing therefrom by thedeletion of 44 minus A amino acids from the amino terminal end, byreplacement of native amino acids at positions 3, 8, 25 by Asn and Asp,by replacement of the methionine residue at position 27 with norleucine(Nle), and by replacement of NH₂ by OH for some of the hGRF analogs.Obviously, GRF analogs wherein X, Y, and Z correspond to the nativesequence, Asp, Asn, and Asp respectively, are not included within thescope of the present invention.

Preferably, the hGRF analogs of the present invention have the sequence[X³, Y⁸, Z²⁵, Nle²⁷ ]-hGRF(1-31)--NH₂, wherein X, Y, and Z are selectedfrom the group consisting of Asn and Asp. The sequence for the preferredhGRF analogs of the present invention expressed using the 3 letter codefor the amino acid is:H-Tyr-Ala-X-Ala-Ile-Phe-Thr-Y-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Z-Ile-Nle-Ser-Arg-Gln-Gln-NH₂,wherein X, Y, and Z are defined as above.

The following peptide sequences, using the shorthand notation, depictexamples of the hGRF analogs of the present invention:

[Asn³, Nle²⁷ ]-hGRF(1-31)--NH₂ ;

[Asp⁸, Nle²⁷ ]-hGRF(1-31)--NH₂ ;

[Asn²⁵, Nle²⁷ ]-hGRF(1-31)--NH₂ ;

[Asn³, Asn²⁵, Nle²⁷ ]-hGRF(1-31)--NH₂ ;

[Asp⁸, Asn²⁵, Nle²⁷ ]-hGRF(1-31)--NH₂ ;

[Asn³, Asp⁸, Nle²⁷ ]-hGRF(1-31)--NH₂ ; and

[Asn³, Asp⁸, Asn²⁵, Nle²⁷ ]-hGRF(1-31)--NH₂.

Each of the hGRF analogs of the present invention are defined to includepharmaceutically acceptable non-toxic acid addition salts and/or apharmaceutically acceptable non-toxic carbolated acid salts.

The term "pharmaceutically acceptable non-toxic acid addition salts"encompasses both organic and inorganic acid addition salts including,for example, those prepared from acids such as hydrochloric,hydrofluoric, sulfuric, sulfonic, tartaric, fumaric, hydrobromic,glycolic, citric, maleic, phosphoric, succinic, acetic, nitric, benzoic,ascorbic, p-toluenesulfonic, benzenesulfonic, naphthalenesulfonic,propionic, and the like. Preferably, the acid addition salts are thoseprepared from hydrochloric acid, acetic acid, or succinic acid. Any ofthe above salts is prepared by conventional methods well known toskilled artisans.

The term "carbolated acid salts" includes, for example, ammonium, alkalimetal salts such as sodium, potassium, and lithium, and the like.

The hGRF analogs of the present invention can be synthesized by any of avariety of recognized peptide synthesis techniques including classical(solution) methods and solid phase methods, with solid phase synthesisbeing preferred.

Solid phase techniques in which the C-terminal amino acid of thesequence is attached to an insoluble support followed by sequentialaddition of the remaining amino acids in the sequence is the preferredmethod for preparing the hGRF analogs of the present invention.Techniques for solid phase synthesis are described by Barany andMerrifield, Solid-Phase Peptide Synthesis; In "The Peptides: Analysis,Synthesis, Biology. Volume 2: Special Methods in Peptide Synthesis, PartA"; Gross and Meienhofer, J. Eds.; Academic Press: New York, 1980; pp.3-284: and J. Stewart et al., Solid Phase Peptide Synthesis, 2ndEdition, Pierce Chemical Co., Rockford, Ill. 1984.

Solid phase synthesis is commenced from the C-terminal end of thepeptide by coupling a protected amino acid to a suitable resin. Astarting material can be prepared by attaching an amino-protected aminoacid via a benzyl ester linkage to a chloromethylated resin or ahydroxymethyl resin or via an amide bond to a benzhydrylamine (BHA)resin or p-methylbenzhydrylamine (MBHA) resin. The resins are availablecommercially and their preparation is known by one of ordinary skill inthe art.

The acid form of peptides may be prepared by the solid phase peptidesynthesis procedure using a benzyl ester resin as a solid support. Thecorresponding amides may be produced by using benzhydrylamine ormethylbenzhydrylamine resin as the solid support for solid phase peptidesynthesis. Those skilled in the art will recognize that when the BHA orMBHA resin is used, treatment with anhydrous HF to remove thepolypeptide from the solid support results in a polypeptide having aterminal amide group.

It should be recognized that the α-amino group of each amino acidemployed in the peptide synthesis must be protected during the couplingreaction to prevent side reactions involving the reactive α-aminofunction. It should also be recognized that certain amino acids containreactive side-chain functional groups (e.g. sulfhydryl, amino, carboxyl,and hydroxyl), and that such functional groups must also be protectedwith suitable protecting groups which will prevent a chemical reactionsfrom occurring at that site both during the initial and subsequentcoupling steps. Suitable protecting groups, known in the art, aredescribed in E. Gross & J. Meienhofer, The Peptides: Analysis,Synthesis, Biology, Volume 3: Protection of Functional Groups in PeptideSynthesis, Academic Press, New York, N.Y., 1981.

In selecting a particular protecting group, certain conditions must beobserved. An α-amino protecting group (1) must render the α-aminofunction inert under the conditions employed in the coupling reaction,(2) must be readily removable after the coupling reaction underconditions that will not remove side chain protecting groups and willnot alter the structure of the peptide fragment, and (3) must eliminatethe possibility of racemization upon activation immediately prior tocoupling. A side chain protecting group (1) must render the side chainfunctional group inert under the conditions employed in the couplingreaction, (2) must be stable under the conditions employed in removingthe α-amino protecting group, and (3) must be readily removable uponcompletion of the desired amino acid sequence under reaction conditionsthat will not alter the structure of the peptide chain.

It will be apparent to those skilled in the art that the protectinggroups known to be useful for peptide synthesis will vary in reactivityto the agents employed for their removal. For example, certainprotecting groups, such as triphenylmethyl and2-(p-biphenylyl)isopropyloxycarbonyl are very labile and can be cleavedunder mild acid conditions. Other protecting groups, such ast-butyloxycarbonyl (Boc), t-amyloxycarbonyl, adamantyloxycarbonyl, andp-methoxybenzyloxycarbonyl, are less labile and require moderatelystrong acids, such as trifluoroacetic, hydrochloric, or borontrifluoride in acetic acid, for their removal. Still other protectinggroups, such as benzyloxycarbonyl (Cbz or Z), halobenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, cycloalkyloxycarbonyl, andisopropyloxycarbonyl, are even less labile and require stronger acids,such as hydrogen fluoride, hydrogen bromide, or boron trifluoroacetatein trifluoroacetic acid, for their removal.

Illustrative examples of amino acid protecting groups include: (1) Foran α-amino group, protection may include (a) aromatic urethane-typegroups, such as fluorenylmethyloxycarbonyl (Fmoc), Cbz, and substitutedbenzyloxycarbonyl, such as, for example, p-chlorobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, andp-methoxybenzyloxycarbonyl, o-chlorobenzyloxycarbonyl,2,4-dichlorobenzyloxycarbonyl, 2,6-dichlorobenzyloxycarbonyl, and thelike; (b) aliphatic urethane-type groups such as Boc, t-amyloxycarbonyl,isopropyloxycarbonyl, 2-(p-biphenylyl)isopropyloxycarbonyl,allyloxycarbonyl, and the like; (c) cycloalkyl urethane-type groups suchas cyclopentyloxycarbonyl, cyclohexyloxycarbonyl,cycloheptyloxycarbonyl, adamantyloxycarbonyl, and the like. Thepreferred α-amino protecting group is t-butyloxycarbonyl (Boc).

(2) For the side chain amino group present in Lys, protection may be byany of the groups mentioned hereinabove for protection of an α-aminogroup. Typical groups include, for example, Boc,p-chlorobenzyloxycarbonyl, p-bromobenzyloxycarbonyl,o-chlorobenzyloxycarbonyl (2-ClZ), 2,6-dichlorobenzyloxycarbonyl,2,4-dichlorobenzyloxycarbonyl, o-bromobenzyloxycarbonyl,p-nitrobenzyloxycarbonyl t-butyloxycarbonyl, isopropyloxycarbonyl,t-amyloxycarbonyl, cyclopentyloxycarbonyl, cyclohexyloxycarbonyl,cyclohepthyloxycarbonyl, adamantyloxycarbonyl, p-toluenesulfonyl, andthe like. The preferred side chain amino protecting group iso-chlorobenzyloxycarbonyl (2-ClZ).

(3) For the guanidino group of Arg, protection may be by nitro, tosyl(Tos), Cbz, adamantyloxycarbonyl, and Boc. The preferred protectinggroup is Tos.

(4) For the hydroxyl group of Ser, Thr, or Tyr, protection may be, forexample, by C₁ -C₄ alkyl, such as methyl, ethyl, and t-butyl; benzyl(Bzl); substituted benzyl, such as p-methoxybenzyl; p-nitrobenzyl,p-chlorobenzyl, o-chlorobenzyl, and 2,6-dichlorobenzyl. The preferredaliphatic hydroxyl protecting group for Ser and Thr is benzyl (Bzl),while the Tyr aromatic hydroxyl is most commonly protected as the2,6-dichlorobenzyl ether (Cl₂ -Bzl).

(5) For the carboxyl group of Asp or Glu, protection may be, forexample, by esterification using groups such as benzyl, t-butyl,cyclohexyl, cyclopentyl, and the like. The preferred groups are benzyl(Bzl) and cyclohexyl (cHex).

The amino acids are coupled to the peptide chain using techniqueswell-known in the art for the formation of peptide bonds. One methodinvolves converting the amino acid to a derivative that will render thecarboxyl group more susceptible to reaction with the free N-terminalamino group of the peptide fragment. For example, the amino acid can beconverted to a mixed anhydride by reaction of a protected amino acidwith ethyl chloroformate, phenyl chloroformate, sec-butyl chloroformate,isobutyl chloroformate, pivaloyl chloride, or like acid chlorides.Alternatively, the amino acid can be converted to an active ester suchas a 2,4,5-trichlorophenyl ester, a pentachlorophenyl ester, ap-nitrophenyl ester, a N-hydroxysuccinimide ester, or an ester formedfrom 1-hydroxybenzotriazole.

Another coupling method involves use of a suitable coupling agent suchas N,N'-dicyclohexylcarbodimide (DCC) or N,N'-diisopropylcarbodiimide(DIPCDI). Other appropriate coupling agents, apparent to those skilledin the art, are disclosed in E. Gross & J. Meienhofer, The Peptides:Analysis, Structure, Biology. Vol. 1: Major Methods of Peptide BondFormation, Academic Press, New York, 1979.

The C-terminal amino acid, e.g. Gln, is protected at the Nα-aminoposition by an appropriately selected protecting group, in the case ofGln by t-butyloxycarbonyl (Boc). The Boc-Gln-OH can be first coupled tothe benzhydrylamine resin using isopropylcarbodiimide at about 25° C.for 2 hours with stirring. Following the coupling of the Boc protectedamino acid to the resin support, the α-amino protecting group isremoved, using trifluoroacetic acid (TFA) in methylene chloride or TFAalone. The deprotection is carried out at a temperature between about 0°C. and room temperature.

After removal of the α-amino protecting group, the remainingBoc-protected amino acids are coupled stepwise in the desired order oras an alternative to adding each amino acid separately in the synthesis,some may be coupled to one another prior to addition to the solid phasesynthesizer. The selection of an appropriate coupling reagent is knownto one of ordinary skill in the art. Particularly suitable isdiisopropylcarbodiimide (DIPCDI).

Each protected amino acid or amino acid sequence is introduced into thesolid phase reactor in excess, and the coupling may be carried out in amedium of dimethylformamide (DMF) or methylene chloride (CH₂ Cl₂) ormixtures thereof. In cases where incomplete coupling occurs, thecoupling procedure is repeated before removal of the Nα-amino protectinggroup prior to the coupling of the next amino acid. The success of thecoupling reaction at each stage of synthesis may be monitored. Apreferred method of monitoring the synthesis is by the ninhydrinreaction. The coupling reactions can be performed automatically usingwell known methods, for example using a Biosearch 9500 PeptideSynthesizer.

Cleavage of the peptide from the resin can be effected using procedureswell known in peptide chemistry. Reaction with hydrogen fluoride in thepresence of anisole and dimethylsulfide at 0° for 1 hour willsimultaneously remove the side chain protecting groups and release thepeptide from the resin.

Purification of the polypeptides of the invention can be effected usingprocedures well known in peptide chemistry. The subject polypeptides maybe purified using preparative HPLC; however, other known chromatographicprocedures well known to skilled artisans such as gel permeation, ionexchange and partition chromatography or countercurrent distribution canalso be employed.

The hGRF analogs of the present invention stimulate the release of GHand therefore have many uses. The present compounds may be used, forexample, in treating primary dwarfism; short stature; wound healing;bone wasting diseases, such as osteoporosis; general catabolic statesdue to illness, trauma, or surgery; fracture healing; and the like. Inaddition, the hGRF analogs of the present invention may be used topromote growth in animals such as cattle, swine, sheep, poultry, and thelike.

According to the present invention, a method for stimulating the releaseof GH in animals comprises administering to the animals an amount of thehGRF analogs of the present invention sufficient to stimulate therelease of GH.

The hGRF analogs of the present invention can be administered as thecompound or as a pharmaceutically acceptable salt of the compound. ThehGRF analogs can be administered alone, in combination, or incombination with pharmaceutically acceptable carriers such as variousdiluents and vehicles. The carrier can be any biocompatible and hGRFcompatible carrier. Most preferably, the hGRF analogs are mixedindividually or in combination with pharmaceutically acceptable carriersto form compositions which allows for easy dosage preparation.

Doses of the hGRF analogs of the present invention are administered tothe recipient for a period during which stimulation of the release of GHis desired. The amount of hGRF analog administered may vary dependingupon the particular type of animal, the maturity of the animal, the sizeof the animal, and whether the dose is to act therapeutically orprophylactically. Generally, the hGRF analogs are administered to theanimal according to the present invention in dosages from about 0.05-100μg/kg of body weight/day (μg/kg/day), preferably from about 0.5-50μg/kg/day.

The hGRF analogs according to the present invention can be administeredto the animals in any acceptable manner including nasally, orally, byinjection, using an implant, and the like. Injections and implants arepreferred because they permit precise control of the timing and dosagelevels used for administration. The hGRF analogs according to thepresent invention are preferably administered parenterally. As usedherein, parenteral administration means administration by intravenous,intramuscular, subcutaneous, or intraperitoneal injection, or bysubcutaneous implant.

When administering the hGRF analogs of the present inventionparenterally, the pharmaceutical formulations suitable for injectioninclude sterile aqueous solutions or dispersions and sterile powders forreconstitution into sterile injectable solutions or dispersions. Thecarrier can be a solvent or dispersing medium containing, for example,water, ethanol, polyol (for example glycerol, propylene glycol, liquidpolyethylene glycol, and the like), suitable mixtures thereof, andvegetable oils. Proper fluidity can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Nonaqueous vehicles such as cottonseed oil, sesame oil, olive oil,soybean oil, corn oil, sunflower oil, or peanut oil and esters such asisopropyl myristate may also be used as solvent systems for compoundcompositions. Additionally, various additives which enhance thestability, sterility, and isotonicity of the compositions includingantimicrobial preservatives, antioxidants, chelating agents, and bufferscan be added. Prevention of the action of microorganisms can be ensuredby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, and the like. In many cases, it willbe desirable to include isotonic agents, for example, sugars, sodiumchloride, and the like. Prolonged absorption of the injectablepharmaceutical form can be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin. Any vehicle,diluent, or additive used would, however, have to be compatible with thecompounds according to the present invention.

Sterile injectable solutions can be prepared by incorporating the hGRFanalogs of the present invention in the required amount of theappropriate solvent with various of the other ingredients, as desired.

The hGRF analogs can be administered to the animals in an injectableformulation containing any biocompatible and compound compatible carriersuch as various vehicles, adjuvants, additives, and diluents.

The hGRF analogs are added to the carrier in amounts sufficient tosupply from about 0.05-100 μg/kg/day to the animal when injected.Preferably, the hGRF analogs are added to the carrier in amountssufficient to supply from about 0.5-50 μg/kg/day to the animal.

The hGRF analogs according to the present invention can be administeredparenterally to the animals in the form of slow-release subcutaneousimplants or targeted delivery systems such as polymer matrices,liposomes, and microspheres. An implant suitable for use in the presentinvention can take the form of a pellet which slowly dissolves afterbeing implanted or a biocompatible and animal compatible delivery modulewell known to those skilled in the art. Such well known dosage forms andmodules are designed such that the active ingredients are slowlyreleased over a period of several days to several weeks. Examples ofwell known implants and modules useful in the present invention include:U.S. Pat. No. 4,487,603 discloses an implantable micro-infusion pump fordispensing medication at a controlled rate. U.S. Pat. No. 4,486,194discloses a therapeutic device for administering medicants through theskin. U.S. Pat. No. 4,447,233 discloses a medication infusion pump fordelivering medication at a precise infusion rate. U.S. Pat. No.4,447,224 discloses a variable flow implantable infusion apparatus forcontinuous drug delivery. U.S. Pat. No. 4,439,196 discloses an osmoticdrug delivery system having multi-chamber compartments. U.S. Pat. No.4,475,196 discloses an osmotic drug delivery system. These patents areincorporated herein by reference. Many other such implants, deliverysystems, and modules are well known to those skilled in the art.

The implant, pellet, module, or other similar delivery system accordingto the present invention is designed to deliver the hGRF analogs inamounts from about 0.05-100 μg/kg/day, preferably from about 0.5-50μg/kg/day.

The hGRF analogs according to the present invention can be administeredorally to the animal. Conventional methods such as administering thecompounds in tablets, suspensions, solutions, emulsions, capsules,powders, syrups, feed compositions, and the like are usable but notpreferred since the biological activity of the peptide is oftendestroyed in the stomach. Known techniques which deliver the peptideorally and retain the biological activity of the peptide are preferred.

According to the present invention, one composition for stimulating therelease of GH in animals comprises a pharmaceutically acceptable carrierand an amount of a hGRF analog sufficient to stimulate the release of GHadmixed with the carrier. The compositions of the present inventioncontains the hGRF analogs in amounts sufficient to supply from about0.05-100 μg/kg of body weight/day (μg/kg/day), preferably from about0.5-50 μg/kg/day.

The composition can be in a form suitable for parenteral administration,typically suspensions, solutions, emulsions, injectable formulations,implants, and the like.

The hGRF analogs according to the present invention can be administeredto the animals in a composition comprising an implant or injectableformulation containing any biocompatible and hGRF analog compatiblecarrier such as various vehicles, adjuvants, additives, and diluents.

Preferably, the composition according to the present invention is (1) animplant pellet comprising a biocompatible and hGRF analogs-compatibleimplant material and an amount of the hGRF analog sufficient tostimulate the release of GH, or (2) an injectable formulation comprisinga biocompatible and hGRF analogs-compatible carrier and an amount of thehGRF analog sufficient to stimulate the release of GH.

It is especially advantageous to formulate the hGRF analogs of thepresent invention in unit dosage form for ease of administration anduniformity of dosage. Unit dosage form as used herein refers to aphysically discrete units suited as unitary dosages for the subject tobe treated. Each unit contains a predetermined quantity of the compoundcalculated to produce the desired therapeutic effect in association withthe pharmaceutically acceptable carrier. The specific unit dosage formis dictated by and directly dependent upon (a) the uniquecharacteristics of the particular composition and (b) the particulartherapeutic or prophylactic effect to be achieved.

Any animal species in need of prophylactic or therapeutic treatmentusing GH can be administered the hGRF analogs and compositions accordingto the present invention. Human, bovine, porcine, canine, feline,equine, avian, and ovine are preferred, with livestock and poultry suchas cattle, swine, sheep, chickens, and turkeys being most preferred.

The invention having been generally described, the following examplesare given as particular embodiments of the invention and to demonstratethe practice and advantages thereof. It is understood that the examplesare given by way of illustration and are not intended to limit thespecification or the claims to follow in any manner.

The following abbreviations are used in examples to indicate variousprotecting groups and reagents; Boc=t-butyloxycarbonyl,Z=benzyloxycarbonyl, 2-ClZ=2-chlorobenzyloxycarbonyl, Bzl=benzyl, Cl₂-Bzl=2,6-dichlorobenzyl, Tos=p-toluenesulfonyl,DIPCDI=diisopropylcarbodiimide, MHBA=p-methylbenzlhydrylamine,DMF=dimethylformamide, TFA=trifluoroacetic acid, CH₂ CH₂ =methylenechloride, and DIPEA=diisopropylethylamine.

The analogs of this invention were prepared by sequential coupling ofamino acids using a commercially available automated solid phase peptidesynthesizer (Biosearch 9500 Peptide Synthesizer). Nα-Boc-amino acidswere used in the synthesis.

Trifunctional amino acids were protected as Nα-Boc-Lys(2-ClZ),Nα-Boc-Asp(Bzl), Nα-Boc-Glu(Bzl), Nα-Boc-Ser(Bzl), Nα-Boc-Thr(Bzl),Nα-Boc-Tyr(Cl₂ -Bzl) and Nα-Boc-Arg(Tos). Protected amino acids can bepurchased from Biosearch, Peptides International, PeninsulaLaboratories, or Bachem.

EXAMPLE 1 Preparation of [Asn³, Nle²⁷ ]-hGRF(1-31)--NH₂

The synthesis of [Asn³, Nle²⁷ ]-hGRF(1-31)--NH₂ is conducted in astepwise manner using classical solid phase techniques (Barany, G.;Merrifield, R. B. Solid-Phase Peptide Synthesis. In "The Peptides:Analysis, Synthesis, Biology, Volume 2: Special Methods in PeptideSynthesis, Part A"; Gross, E., Meienhofer, J. Eds.; Academic Press: NewYork, 1980; pp. 3-284). Solvents were HPLC grade, DMF was purged withnitrogen for 15-30 minutes prior to use. Couplings were done bytreatment with 6.7 molar excess of the amino acid in the presence of anequivalent amount of DIPCDI for 2 hours in a 1:1 mixture of CH₂ Cl₂ andDMF. Asn and Gln were coupled in the presence of 1.5 equivalents of1-hydroxybenzotriazole. The following amino acids were double coupled:Asn, Gln, Ile, Val, and Nle. Unreacted amino groups were capped bytreatment with 0.3M 1-acetylimidazole in DMF before proceeding with thenext synthetic cycle. The Boc groups were removed by treatment with asolution of 45 % TFA and 2.5% anisole in dichloromethane.

Boc-Gln-OH was coupled to 200 g MBHA-resin containing approximately 0.3mmol of amino groups per gram of resin. The procedure for coupling andcapping is carried out in accordance with Schedule A.

    ______________________________________                                        Schedule A                                                                    Reagent              Mixing Time (Min:Sec)                                    ______________________________________                                        1.  CH.sub.2 Cl.sub.2 (5 times)                                                                        0:15                                                 2.  CH.sub.2 Cl.sub.2 /DMF (1:1)                                                                       1:30                                                 3.  DIPCDI in CH.sub.2 Cl.sub.2 + Boc-amino                                                            110:00                                                   acid in DMF                                                               4.  CH.sub.2 Cl.sub.2 /DMF (1:1) (2 times)                                                             0:15                                                 5.  CH.sub.2 Cl.sub.2 (5 times)                                                                        0:15                                                 6.  DMF                  0:15                                                 7.  1-Acetylimidazole in DMF                                                                           60:00                                                8.  DMF (2 times)        0:15                                                 ______________________________________                                    

The procedure for removal of the Boc group (deblocking) after eachcoupling is carried out as set forth in Schedule B.

    ______________________________________                                        Schedule B                                                                    Reagent              Mixing Time (Min:Sec)                                    ______________________________________                                        1.  CH.sub.2 Cl.sub.2 (4 times)                                                                        0:15                                                 2.  40% TFA/2.5% Anisole in CH.sub.2 Cl.sub.2                                                          1:00                                                 3.  40% TFA/2.5% Anisole in CH.sub.2 Cl.sub.2                                                          20:00                                                4.  CH.sub.2 Cl.sub.2 (2 times)                                                                        0:15                                                 5.  50% DMF/50% CH.sub.2 Cl.sub.2 (3 times)                                                            0:15                                                 6.  CH.sub.2 Cl.sub.2 (2 times)                                                                        0:15                                                 7.  10% DIPEA in CH.sub.2 Cl.sub.2 (3 times)                                                           0:15                                                 8.  CH.sub.2 Cl.sub.2 (5 times)                                                                        0:15                                                 ______________________________________                                    

After completing the sequential addition of all of the amino acids, theresin bound peptide is dried under vacuum to provide 3.73 g of crude,resin-bound peptide. A 2.01 g portion of the resin-bound peptide wascleaved from the resin by treatment with 10 mL of anhydrous hydrogenfluoride (distilled from cobalt (III) fluoride), 1 mL of anisole and 1ml of dimethyl sulfide per gram of resin for 30-45 min at 0° C. The HFand volatile organics were removed by vacuum distillation and theresidue dried overnight in a vacuum desiccator. The free peptide wasprecipitated with diethyl ether, extracted with 50% acetic acid,concentrated under reduced pressure and lyophilized to provide 925 mg ofcrude peptide.

The crude peptide was purified by semi-preparative HPLC. The crudepeptide was dissolved into water containing 0.1% TFA and approximately10-50 mg was loaded onto a 1.0×25 cm Vydac C₁₈, 5μ, 300A HPLC column(catalog number 218TP510). The column was eluted at a flow rate of2.0-2.5 ml/min with a solvent system consisting of water containing 0.1%TFA (Solvent A) and 80% acetonitrite in water containing 0.1% TFA(Solvent B) in a linear gradient from 30% solvent B to 60% solvent Bover 60 minutes. The eluent was monitored using a UV detector set at 220nm and the desired peak was collected. The fractions from several suchruns were combined, lyophilized, and repurified as above when necessary.A total of 3.1 mg of pure peptide was obtained from 300 mg of the crudeproduct.

The final product was shown to be homogeneous by analytical HPLC in twodifferent solvent systems; thin layer chromatography (TLC); and aminoacid analysis.

EXAMPLE 2 Preparation of [Asp⁸,Nle²⁷ ]-hGRF(1-31)--NH₂

The synthesis of [Asp⁸,Nle²⁷ ]-hGRF(1-31)--NH₂ was carried out asdescribed in Example 1 beginning with 2.00 g of MHBA-resin. The yield ofcrude, resin-bound peptide after completion of the synthesis cycles was3.01 g. A 2.00 g portion of the resin-bound peptide was cleaved from theresin as described in Example 1 to provide 440 mg of the crude analog.HPLC purification of 150 mg of the crude peptide as set forth in Example1 provided 3.0 mg of the pure analog. The peptide was judged to behomogeneous by analytical HPLC; TLC; and amino acid analyses.

EXAMPLE 3 Preparation of [Asn²⁵,Nle²⁷ ]-hGRF(1-31)--NH₂

The synthesis of [Asn²⁵,Nle²⁷ ]-hGRF(1-31)--NH₂ was carried out asdescribed in Example 1 beginning with 2.13 g of MBHA-resin. The yield ofcrude, resin-bound peptide after completion of the synthesis cycles was3.34 g. A 1.57 g portion of the resin-bound peptide was cleaved from theresin as described in Example 1 to provide 250 mg of the crude analog.HPLC purification of 250 mg of the crude peptide as set forth in Example1 provided 5.2 mg of the pure analog. The peptide was judged to behomogeneous by analytical HPLC; TLC; and amino acid analyses.

EXAMPLE 4 Preparation of [Asn³,Asp⁸,Nle²⁷ ]-hGRF(1-31)--NH₂

The synthesis of [Asn²⁵,Nle²⁷ ]-hGRF(1-31)--NH₂ was carried out asdescribed in Example 1 beginning with 1.04 g of MBHA-resin. The yield ofcrude, resin-bound peptide after completion of the synthesis cycles was1.61 g. A 1.60 g portion of the resin-bound peptide was cleaved from theresin as described in Example 1 to provide 681 mg of the crude analog.HPLC purification of 25.0 mg of the crude peptide as set forth inExample 1 provided 1.0 mg of the pure analog. The peptide was judged tobe homogeneous by analytical HPLC; TLC; and amino acid analyses.

EXAMPLE 5 Preparation of [Asn³,Asn²⁵,Nle²⁷ ]-hGRF (1-31)--NH₂

The synthesis of [Asn³,Asn²⁵,Nle²⁷ ]-hGRF (1-31)--NH₂ was carried out asdescribed in Example 1 beginning with 2.00 g of MBHA-resin. The yield ofcrude, resin-bound peptide after completion of the synthesis cycles was3.88 g. A 2.01 g portion of the resin-bound peptide was cleaved from theresin as described in Example 1 to provide 585 mg of the crude analog.HPLC purification of 150 mg of the crude peptide as set forth in Example1 provided 5.0 mg of the pure analog. The peptide was judged to behomogeneous by analytical HPLC; TLC; and amino acid analyses.

EXAMPLE 6 Preparation of [Asp⁸,Asn²⁵,Nle²⁷ ]-hGRF(1-31)--NH₂

The synthesis of [Asp⁸,Asn²⁵,Nle²⁷ ]-hGRF(1-31)--NH₂ was carried out asdescribed in Example 1 beginning with 2.51 g of MBHA-resin. The yield ofcrude, resin-bound peptide after completion of the synthesis cycles was4.30 g. A 2.13 g portion of the resin-bound peptide was cleaved from theresin as described in Example 1 to provide 850 mg of the crude analog.HPLC purification of 310 mg of the crude peptide as set forth in Example1 provided 8.0 mg of the pure analog. The peptide was judged to behomogeneous by analytical HPLC; TLC; and amino acid analyses.

EXAMPLE 7 Preparation of [Asn³,Asp⁸,Asn²⁵,Nle²⁷ ]-hGRF(1-31)--NH₂

The synthesis of [Asn³,Asp⁸,Asn²⁵,Nle²⁷ ]-hGRF(1-31)--NH₂ was carriedout as described in Example 1 beginning with 2.00 g of MBHA-resin. Theyield of crude, resin-bound peptide after completion of the synthesiscycles was 3.54 g. A 1.51 g portion of the resin-bound was cleaved fromthe resin as described in Example 1 to provide 625 mg of the crudeanalog. HPLC purification of 185 mg of the crude peptide as set forth inExample 1 provided 5.0 mg of the pure analog. The peptide was judged tobe homogeneous by analytical HPLC; TLC; and amino acid analyses.

EXAMPLE 8 Bioassay of hGRF Analogs

The biological activity of the synthesized hGRF analogs of the presentinvention was compared with that of synthetic hGRF(1-44)--NH₂ which iscomparable to the natural hGRF(1-44)--NH₂. Biological activity of thesynthetic hGRF(1-44)--NH₂ was identical to the natural hGRF(1-44)--NH₂which was isolated from a human pancreatic tumor of an individualsuffering from acromegaly (Salk Institute standard hGRF-NH₂(NL-A-10).4). The assay for biological activity, which is based on theability to stimulate release of growth hormone in rat pituitary cells intissue culture, has been described in detail (Culler, M. D.; Kenjo, T.:Obara, N.; Arimuar, A., Stimulation of cAMP. Accumulation by HumanPancreatic GH-Releasing Factor(1-44). Am. J. Physiol. 1984, 247(Endocrinol.Metab. 10). E609-E615.

ALLFIT, the 4-parameter logistic curve fitting program available fromthe Biomedical Computer Technology Information Center, VanderbiltMedical Center, Nashville Tenn., was used to determine ED₅₀ and maximalstimulated values from the data. In several instances, the amount of GHreleased reached a maximum value and decreased with further increasingdoses of the hGRF analogs. To simplify the data analysis, these valueswere not used in the calculations. The actual data points are shown inTable 1. The calculated maximal responses, the doses of the analogrequired to release the amount of GH released at the ED₅₀ of thestandard (hGRF) and the potencies of the analogs relative to hGRF, aregiven in Table 2. Generally all novel analogs were fully active and wereable to stimulate the release of growth hormone with maximal values ofGH release being within 80% of the value for natural hGRF. There was awide distribution of potencies as seen by the shift in the dose-responsecurves of the analogs.

Referring to Table 1, GRF analogs [Nle²⁷ ]-hGRF(1-31)NH₂, [Asn³, Nle²⁷]-hGRF(1-31)--NH₂, [Asp⁸, Nle²⁷ ]-hGRF(1-31)--NH₂, [Asn²⁵, Nle²⁷]-hGRF(1-31)NH₂, [Asn³, Asn²⁵, Nle²⁷ ]-hGRF(1-31)--NH₂, and [Asp⁸,Asn²⁵, Nle²⁷ ]-hGRF(1-31)--NH₂ induced GH release in dosages comparableto those for native hGRF while analogs [Asn³, Asp⁸, Nle²⁷]-hGRF(1-31)--NH₂ ; and [Asn³, Asp⁸, Asn²⁵, Nle²⁷ ]-hGRF(1-31)--NH₂ didnot induce GH release at dosages comparable to native hGRF.Surprisingly, the data indicates the some analogs of the presentinvention having substitutions at positions 3, 8, and 25 retain highlevels of bioactivity while others retain only low levels ofbioactivity. While not wishing to be bound by theory, it is believedthat the nature of the residues at these positions is important fordetermining receptor binding affinities and therefore bioactivity.

Referring to Table 2, the data suprisingly shows that the potency of theanalogs of the present invention varies greatly. Potency of 82 and 76%were found for analogs [Asp⁸,Nle²⁷ ]-hGRF(1-31)--NH₂ and[Asp⁸,Asn²⁵,Nle²⁷ ]-hGRF(1-31)--NH₂, respectively. In contrast, potencyof only 3 and 5% was found for analogs, [Asn³,Asp⁸,Nle²⁷]-hGRF(1-31)--NH₂ and [Asn³,Asp⁸,Asn²⁵,Nle²⁷ ]-hGRF(1-31)--NH₂,respectively. The analog [Asn²⁵,Nle²⁷ ]-hGRF(1-31)--NH₂ was 2.7 timesmore potent than hGRF(1-44)--NH₂ on a molar basis.

Obviously many modifications and variations of the present invention arepossible in light of the above teachings. It is therefore to beunderstood that within the scope of the appended claims the inventionmay be practiced otherwise than as specifically described.

                  TABLE 1                                                         ______________________________________                                        GH Release from Cultured Pituitary Cells                                      Stimulated by the hGRF Analogs                                                                         GH Released                                          Analog         Dose (M)  (μg/mL:mean ± (S.E.))                          ______________________________________                                        --             --        0.69 (0.05)                                          hGRF           1 × 10.sup.-11                                                                    0.73 (0.03)                                                         1 × 10.sup.-10                                                                    1.16 (0.04)                                                         1 × 10.sup.-9                                                                     2.84 (0.12)                                                         1 × 10.sup.-8                                                                     3.44 (0.23)                                                         1 × 10.sup.-7                                                                     3.17 (0.12)                                                         1 × 10.sup.-6                                                                     2.81 (0.13)                                          [Nle.sup.27 ]- 1 × 10.sup.-11                                                                    0.85 (0.03)                                                         1 × 10.sup.-10                                                                    1.93 (0.10)                                                         1 × 10.sup.-9                                                                     3.16 (0.12)                                                         1 × 10.sup.-8                                                                     3.11 (0.23)                                                         1 × 10.sup.-7                                                                     2.78 (0.09)                                                         1 × 10.sup.-6                                                                     2.89 (0.08)                                          [Asn.sup.3, Nle.sup.27 ]-                                                                    1 × 10.sup.-11                                                                    0.67 (0.08)                                                         1 × 10.sup.-10                                                                    0.97 (0.01)                                                         1 × 10.sup.-9                                                                     2.40 (0.06)                                                         1 × 10.sup.-8                                                                     2.90 (0.12)                                                         1 × 3.05 (0.09)                                                         1 × 10.sup.-6                                                                     3.22 (0.16)                                          [Asp.sup.8, Nle.sup.27 ]-                                                                    1 × 10.sup.-11                                                                    0.61 (0.03)                                                         1 × 10.sup.-10                                                                    1.18 (0.10)                                                         1 × 10.sup.-9                                                                     2.69 (0.12)                                                         1 × 10.sup.-8                                                                     3.59 (0.17)                                                         1 × 10.sup.-7                                                                     3.52 (0.22)                                                         1 × 10.sup.-6                                                                     3.17 (0.24)                                          [Asn.sup.25, Nle.sup.27 ]-                                                                   1 × 10.sup.-11                                                                    0.72 (0.05)                                                         1 × 10.sup.-10                                                                    1.84 (0.06)                                                         1 × 10.sup.-9                                                                     3.12 (0.15)                                                         1 × 10.sup.-8                                                                     3.10 (0.12)                                                         1 × 10.sup.-7                                                                     3.22 (0.09)                                                         1 × 10.sup.-6                                                                     3.22 (0.24)                                          [Asn.sup.3, Asp.sup.8, Nle.sup.27 ]-                                                         1 × 10.sup.-11                                                                    0.65 (0.04)                                                         1 × 10.sup.-10                                                                    0.58 (0.04)                                                         1 × 10.sup.-9                                                                     1.04 (0.05)                                                         1 × 10.sup.-8                                                                     2.27 (0.07)                                                         1 × 10.sup.-7                                                                     3.30 (0.27)                                                         1 × 10.sup.-6                                                                     2.74 (0.19)                                          [Asn.sup.3, Asn.sup.25, Nle.sup.27 ]-                                                        1 × 10.sup.-11                                                                    0.65 (0.03)                                                         1 × 10.sup.-10                                                                    0.81 (0.06)                                                         1 × 10.sup.-9                                                                     1.58 (0.07)                                                         1 × 10.sup.-8                                                                     3.21 (0.13)                                                         1 × 10.sup.-7                                                                     3.30 (0.08)                                                         1 × 10.sup.-6                                                                     2.96 (0.11)                                          [Asp.sup.8, Asn.sup.25, Nle.sup.27 ]-                                                        1 × 10.sup.-11                                                                    0.74 (0.09)                                                         1 × 10.sup.-10                                                                    0.96 (0.07)                                                         1 × 10.sup.-9                                                                     2.59 (0.17)                                                         1 × 10.sup.-8                                                                     2.86 (0.08)                                                         1 × 10.sup.-7                                                                     2.93 (0.17)                                                         1 × 10.sup.-6                                                                     2.86 (0.07)                                          [Asn.sup.3, Asp.sup.8, Asn.sup.25, Nle.sup.27 ]-                                             1 × 10.sup.-11                                                                    0.60 (0.02)                                                         1 × 10.sup.-10                                                                    0.61 (0.05)                                                         1 × 10.sup.-9                                                                     0.75 (0.01)                                                         1 × 10.sup.-8                                                                     1.88 (0.07)                                                         1 × 10.sup.-7                                                                     2.94 (0.11)                                                         1 × 10.sup.-6                                                                     3.12 (0.12)                                          ______________________________________                                         S.E. =  standard error                                                   

                  TABLE 2                                                         ______________________________________                                        Bioactivity of the hGRF Analogs from                                          Curves Generated by ALLFIT                                                             Maximum   Relative   Dose Needed                                              GH        Maximum    to Release                                                                             Po-                                    hGRF (1-31)-                                                                           Released  Activity   2.01 μg/ml                                                                          tency.sup.b                            NH.sub.2 Analog                                                                        (μg/mL)                                                                              (% of hGRF)                                                                              of GH.sup.a (nM)                                                                       (%)                                    ______________________________________                                        hGRF (1- 3.31      100        0.32     100                                    44)-NH.sub.2                                                                  [Nle.sup.27 ]                                                                          3.16      95         0.11     291                                    [Asn.sup.3, Nle.sup.27 ]                                                               3.09      93         0.58     55                                     [Asp.sup.8, Nle.sup.27 ]                                                               3.60      109        0.39     82                                     [Asn.sup.25, Nle.sup.27 ]                                                              3.18      96         0.12     267                                    [Asn.sup.3, Asp.sup.8,                                                                 3.53      107        6.74      5                                     Nle.sup.27 ]                                                                  [Asn.sup.3, Asn.sup.25,                                                                3.33      101        1.54     21                                     Nle.sup.27 ]                                                                  [Asp.sup.8, Asn.sup.25,                                                                2.89      87         0.42     76                                     Nle.sup.27 ]                                                                  [Asn.sup.3, Asp.sup.8,                                                                 3.11      94         12.05     3                                     Asn.sup.25, Nle.sup.27 ]                                                      ______________________________________                                         .sup.a ED.sub.50 Value for hGRF                                               .sup. b (ED.sub.50 of hGRF)/(Dose required to give same response as           ED.sub.50 of hGRF).                                                           nM = nanomolar                                                           

I claim: .[.1. A human Growth Hormone-releasing Factor (hGRF) analoghaving the sequence selected from the group consisting [Asp⁸, Asn²⁵,Nle²⁷ ]-hGRF(1-31)--NH₂..].
 5. The method of claim .[.3.]. .Iadd.17.Iaddend.wherein the hGRF analog is administered in dosages of fromabout 0.05-100 μg/kg of body weight/day.
 6. The method of claim .[.3.]..Iadd.17 .Iaddend.wherein the hGRF analog is administered parenterally.7. The method of claim 6 wherein the hGRF analog is administered usingan implant, said implant further comprising:a biocompatible and the hGRFanalog compatible implant material; and an amount of the hGRF analogsufficient to stimulate the release of GH.
 8. The method of claim 6wherein the hGRF analog is administered in an injectable formulation,said injectable formulation further comprising:a biocompatible and thehGRF analog compatible carrier; and an amount of the hGRF analogsufficient to stimulate the release of GH.
 9. The method of claim.[.3.]. .Iadd.17 .Iaddend.wherein said animals are selected from thegroup consisting of cattle and sheep. .[.10. A composition forstimulating the release of growth hormone (GH) in animals, comprising:apharmaceutically acceptable carrier; and an amount of a human growthhormone-releasing factor (hGRF) analog sufficient to stimulate therelease of GH, the hGRF analog having a sequence selected from the groupconsisting of: [Asp⁸, Nle²⁷ ]-hGRF(1-31)--NH₂ ; [Asn²⁵, Nle²⁷]-hGRF(1-31)--NH₂ ; [Asn³, Asn²⁵, Nle²⁷ ]-hGRF(1-31)--NH₂ ; [Asp⁸,Asn²⁵, Nle²⁷ ]-hGRF(1-31)--NH₂ ; [Asn³, Asp⁸, Nle²⁷ ]-hGRF(1-31)--NH₂ ;and [Asn³, Asp⁸, Asn²⁵, Nle²⁷ ]-hGRF(1-31)--NH₂..]. .[.1. Thecomposition of claim 10 wherein the hGRF analog has a sequence selectedfrom the group consisting of:[Asp⁸, Nle²⁷ ]-hGRF(1-31)--NH₂ ; [Asn²⁵Nle²⁷ ]-hGRF(1-31)--NH₂ ; [Asn³, Asn²⁵, Nle²⁷ ]-hGRF(1-31)--NH₂ ; and[Asp⁸, Asn²⁵, Nle²⁷ ]-hGRF(1-31)--NH₂..].
 12. The composition of claim.[.10.]. .Iadd.18 .Iaddend.containing the hGRF analog in dosages of fromabout 0.05-100 μg/kg of body weight/day.
 13. The composition of claim.[.10.]. .Iadd.18 .Iaddend.in a form suitable for parenteraladministration.
 14. The composition of claim 13 in the form of animplant, said implant further comprising:a biocompatible and the hGRFanalog compatible implant material; and an amount of the hGRF analogsufficient to stimulate the release of GH. The composition of claim 13in the form of an injectable formulation, said injectable formulationfurther comprising:a biocompatible and the hGRF analog compatiblecarrier; and an amount of the hGRF analog sufficient to stimulate therelease of GH. .Iadd.16. A Human Growth Hormone-releasing Factor (hGRF)analog having the sequence [Asn²⁵, Nle²⁷ ]-hGRF(1-31)--NH₂. .Iaddend..Iadd.17. A method for stimulating the release of growth hormone (GH) inanimals, comprising: administering to said animals an amount of a humangrowth hormone-releasing factor (hGRF) analog sufficient to stimulatethe release of growth hormone (GH), the hGRF analog having the sequence[Asn²⁵, Nle²⁷ ]-hGRF(1-31)--NH₂. .Iaddend. .Iadd.18. A composition forstimulating the release of growth hormone (GH) in animals, comprising: apharmaceutically acceptable carrier; and an amount of a human growthhormone-releasing factor (hGRF) analog sufficient to stimulate therelease of GH, the hGRF analog having the sequence [Asn²⁵, Nle²⁷]-hGRF(1-31)--NH₂. .Iaddend.